US20230242983A1 - Bioparticle analysis method and bioparticle analysis system - Google Patents

Bioparticle analysis method and bioparticle analysis system Download PDF

Info

Publication number
US20230242983A1
US20230242983A1 US18/012,605 US202118012605A US2023242983A1 US 20230242983 A1 US20230242983 A1 US 20230242983A1 US 202118012605 A US202118012605 A US 202118012605A US 2023242983 A1 US2023242983 A1 US 2023242983A1
Authority
US
United States
Prior art keywords
bioparticle
analysis
molecule
analysis method
linker
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
US18/012,605
Other languages
English (en)
Inventor
Masahiro Matsumoto
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Sony Group Corp
Original Assignee
Sony Group Corp
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Sony Group Corp filed Critical Sony Group Corp
Assigned to Sony Group Corporation reassignment Sony Group Corporation ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: MATSUMOTO, MASAHIRO
Publication of US20230242983A1 publication Critical patent/US20230242983A1/en
Pending legal-status Critical Current

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6816—Hybridisation assays characterised by the detection means
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6869—Methods for sequencing
    • C12Q1/6874—Methods for sequencing involving nucleic acid arrays, e.g. sequencing by hybridisation
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6841—In situ hybridisation
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2458/00—Labels used in chemical analysis of biological material
    • G01N2458/10—Oligonucleotides as tagging agents for labelling antibodies
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
    • G01N33/582—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with fluorescent label

Definitions

  • the present technology relates to a bioparticle analysis method and a bioparticle analysis system. More specifically, the present technology relates to a bioparticle analysis method including a step of isolating a bioparticle into a microspace to analyze components of the bioparticle, and a system that performs the method.
  • a barcoding technique using a DNA sequence As a method for performing single cell analysis, a barcoding technique using a DNA sequence has been proposed. Examples of the barcoding technique include a method using wells and a method using an emulsion.
  • Non-Patent Document 1 An example of the method using wells is described in Non-Patent Document 1 below.
  • Non-Patent Document 2 describes a droplet-based microfluidic protocol for high-throughput analysis and sorting of single cells.
  • the above-described barcoding technique using wells may take time in analyzing cells, for example, because processing for putting one cell into one well takes time. Therefore, it is desirable to improve analysis efficiency in the barcoding technique using wells.
  • the above-described barcoding technique using an emulsion there is a low probability that one cell and one barcode are put into one emulsion particle. Therefore, there has been a demand for improving analysis efficiency in the barcoding technique using an emulsion as well.
  • an object of the present technology is to improve analysis efficiency in single cell analysis using a barcoding technique.
  • the present inventor has found that analysis efficiency can be improved by isolating each cell into a microspace after a barcode is assigned to each cell.
  • the present technology provides a bioparticle analysis method including:
  • a plurality of molecules bound to one bioparticle may have the same barcode sequence.
  • the bioparticle analysis method may further include a disruption step of disrupting the bioparticle in the microspace.
  • the molecule may be dissociated from the bioparticle.
  • the molecule may further include a target substance capturing part, and
  • a target substance constituting the bioparticle or a target substance bound to the bioparticle may be captured by the target substance capturing part.
  • the bioparticle analysis method may further include an analysis step of analyzing the target substance after the disruption step.
  • the barcode sequence may be associated with the target substance.
  • the target substance may have a base sequence
  • sequencing processing may be performed on the base sequence of the target substance.
  • a bioparticle to be released from the surface may be selected on the basis of a label of the bioparticle or a label of the molecule.
  • the linker may be cleaved by chemical stimulation or photic stimulation.
  • the captured state of the bioparticle may be maintained by the bioparticle capturing part.
  • a selected bioparticle may be released from the surface.
  • the microspace may be a space in an emulsion particle or a space in a well.
  • the bioparticle analysis method may further include a determination step of determining whether to isolate a bioparticle released from the surface in the cleavage step into a microspace.
  • the determination may be performed on the basis of light generated by irradiating the bioparticle with light.
  • the bioparticle and the bioparticle capturing part may be bound to each other in a specific or non-specific manner.
  • the capture step may include an incubation step for binding the bioparticle and the bioparticle capturing part to each other.
  • bioparticle analysis system including:
  • a capture kit configured to capture a bioparticle, the capture kit having a surface on which a molecule including a bioparticle capturing part, a barcode sequence, and a cleavable linker is immobilized via the linker;
  • linker cleavage device that cleaves the linker to release the bioparticle from the surface
  • an isolation device that forms or has a microspace into which the bioparticle released from the surface is isolated.
  • the bioparticle analysis system may further include an analysis device that analyzes a target substance constituting the bioparticle or a target substance bound to the bioparticle.
  • FIG. 1 is an example of a flowchart of an analysis method according to the present technology.
  • FIG. 2 is a schematic diagram for explaining an operation in each step included in the analysis method according to the present technology.
  • FIG. 3 is a schematic view illustrating an analysis surface used in the analysis method according to the present technology and molecules immobilized on the surface.
  • FIG. 4 is a schematic diagram for explaining a particle isolation step.
  • FIG. 5 is a schematic diagram for explaining the particle isolation step.
  • FIG. 6 is a diagram illustrating an example of a microchannel used in the particle isolation step.
  • FIG. 7 is a diagram illustrating an example of a nucleic acid-bound antibody.
  • FIG. 8 A is a diagram illustrating an example of a bioparticle sorting device used in the particle isolation step.
  • FIG. 8 B is a schematic view illustrating how emulsion particles are formed and bioparticles are isolated into the formed emulsion particles in a microchip.
  • FIG. 9 is an enlarged view of a particle sorting part included in the bioparticle sorting device.
  • FIG. 10 is a diagram illustrating an example of a block diagram of a control unit included in the bioparticle sorting device.
  • FIG. 11 is an example of a flowchart of processing by the bioparticle sorting device.
  • FIG. 12 A is an enlarged view of the vicinity of a connection channel included in the bioparticle sorting device.
  • FIG. 12 B is an enlarged view of the vicinity of the connection channel included in the bioparticle sorting device.
  • FIG. 13 A is an enlarged view of the vicinity of a connection channel included in the bioparticle sorting device.
  • FIG. 13 B is an enlarged view of the vicinity of the connection channel included in the bioparticle sorting device.
  • FIG. 14 is a schematic view of the bioparticle sorting device to which a collection container is connected.
  • FIG. 15 is a copy of a photograph showing that an emulsion particle was formed by the bioparticle sorting device.
  • FIG. 16 is a diagram illustrating another example of the microchip.
  • a barcoding technique using a DNA sequence has been proposed.
  • This method makes it possible to detect a large number of molecules (e.g., cell surface antigens, intracellular proteins, and mRNAs).
  • one cell and one bar coding bead are put in a microspace, e.g., a well or an emulsion, and then, the cell is lysed, such that a barcode may be assigned to a molecule to be measured.
  • the analysis using this method may be performed, for example, as follows. That is, different barcode molecules are present in respective microspaces. Then, cells are put into the microspaces, and thereafter, the cells are lysed and incubated, such that the barcode molecule is bound to a molecule to be measured in each microspace to form a barcoded molecule to be measured. Then, all the barcoded molecules to be measured are mixed, and sequences of the molecules to be measured are decrypted by an analysis device, e.g., a next-generation sequencing (NGS).
  • NGS next-generation sequencing
  • the analysis method using the next-generation sequencer is capable of detection even in a case where the number of markers is 10 2 or more, for example, because there is no spectral overlap which is a disadvantage of a fluorescence detection method (e.g., flow cytometry or fluorescence imaging).
  • a fluorescence detection method e.g., flow cytometry or fluorescence imaging.
  • Analysis efficiency in single cell analysis using a barcoding technique depends on a probability that one cell and one barcode bead (or gel bead) are encapsulated in one microspace. In addition, the analysis efficiency may decrease due to various factors that are peculiar to an analysis method.
  • a cell sorter is used to place one cell in one well, or a smaller number of cells than the number of wells are seeded in a well plate. Thereafter, single cell barcoding is performed by placing a barcode bead (e.g., gel bead).
  • a barcode bead e.g., gel bead
  • a sample may be taken by a micropipette from a well satisfying the condition that one cell and one barcode bead (or gel bead) are contained in one well, or alternatively, a sample derived from a well that does not satisfy the condition may be excluded.
  • a sample may be taken by a micropipette from a well satisfying the condition that one cell and one barcode bead (or gel bead) are contained in one well, or alternatively, a sample derived from a well that does not satisfy the condition may be excluded.
  • such processing is time consuming and inefficient.
  • analysis is performed by a next-generation sequencer without selectively acquiring a sample capable of single cell analysis, and then, data derived from a sample that is not a single cell is excluded, but this leads to a waste in the analysis.
  • a single cell is sorted on a well plate using a cell sorter on the basis of a detection signal, for example, the number of wells in the well plate is limited, resulting in a low analysis scale and throughput.
  • sorting cannot be performed while a sorting nozzle moves between the wells, the target cells flowing during this time are lost.
  • an emulsion-based method is easier in increasing an analysis scale than the well-based method.
  • a solution containing particles to be captured e.g., a hydrophilic solution
  • a solution immiscible therewith e.g., a hydrophobic solution
  • the hydrophilic solution may be captured in the hydrophobic solution by changing a flow rate of each solution while the solutions flow.
  • an emulsion may be formed in the microchannel, and then, particles and barcode beads may be captured in emulsion particles.
  • the hydrophilic solution is incorporated into the hydrophobic solution to form emulsion particles at a regular timing, and particles and barcode beads incorporated into the emulsion particles are contained in the hydrophilic solution in a diluted state at a predetermined concentration. This is to avoid formation of doublet in which two cells or two barcode beads are captured in one emulsion particle. Accordingly, in many cases, cells or barcode beads are not captured in the emulsion particles, and the emulsion particles are often empty.
  • a chance of success in capturing a cell in an emulsion particle is not high, and it has been said that the chance of success in capturing one cell in one emulsion particle is about 65% at the maximum.
  • a probability that one barcode bead is captured in addition to one cell in one emulsion particle is further reduced.
  • a probability that no, one, or two or more cells are put in one emulsion particle basically follows a Poisson distribution (see Non-Patent Document 2 above).
  • the number of cells in a sample for single cell analysis is small, particularly in a clinical sample from which only 10 4 to 10 5 cells can be obtained or a rare cell sample such as CTC, it is important to efficiently capture one cell and one barcode bead or one gel bead in one emulsion.
  • the emulsion particle in a case where two or more cells are put in one emulsion particle, the emulsion particle is not a target for single cell analysis.
  • analysis processing e.g., next-generation sequencing
  • analysis data thereof is excluded. Therefore, the formation of doublet leads to a waste of analysis.
  • a target cell group to be subjected to single cell analysis may be sorted and purified using a cell sorter or the like.
  • a device called a cell sorter is required in addition to the emulsion forming device, and a workflow called cell sorting is also added. Therefore, costs increase in terms of time and expense.
  • an object of the present technology is to provide a method for improving analysis efficiency in single cell analysis using a barcode sequence.
  • a bioparticle analysis method includes: a capture step of capturing a bioparticle on a surface, on which a molecule including a bioparticle capturing part, a barcode sequence, and a cleavable linker is immobilized via the linker, via the bioparticle capturing part; a cleavage step of cleaving the linker to release the bioparticle from the surface; and an isolation step of isolating the bioparticle into a microspace.
  • the barcode sequence and the bioparticle can be efficiently isolated into one microspace.
  • a target substance contained in the bioparticle can be efficiently bound to the barcode sequence, and furthermore, each bioparticle can be efficiently analyzed. In this way, according to the present technology, efficiency in single cell analysis using a barcode sequence can be improved.
  • the bioparticle analysis method may further include a disruption step of disrupting the bioparticle in the microspace.
  • the bioparticle analysis method may further include an analysis step of analyzing a target substance contained in the bioparticle.
  • the target substance may be, for example, a substance bound to the molecule containing the barcode sequence.
  • the target substance may be a nucleic acid, for example, an RNA or a DNA, particularly an mRNA.
  • FIG. 1 illustrates an example of a flowchart of a bioparticle analysis method according to the present technology.
  • the analysis method according to the present technology may include a preparation step S 101 , a capture step S 102 , a cleavage step S 103 , an isolation step S 104 , a disruption step S 105 , and an analysis step S 106 . These steps will be described below.
  • a surface on which a molecule including a bioparticle capturing part, a barcode sequence, and a cleavable linker is immobilized via the linker is prepared.
  • an analysis substrate e.g., a slide glass
  • a surface 101 on which a plurality of molecules 100 is immobilized is prepared.
  • the molecule 100 includes a bioparticle capturing part, a barcode sequence, and a cleavable linker.
  • the molecule including a bioparticle capturing part, a barcode sequence, and a linker is a molecule used for capturing a target substance, and may also be referred to as a target capturing molecule.
  • the target capturing molecule is a term for referring to a molecule used in the present technology, and may also be used in the present specification to refer to, for example, the molecule after the target substance is captured or the molecule after the linker is cleaved in the cleavage step to be described later.
  • the target capturing molecule may be, for example, either a single molecule or 08 or a complex molecule.
  • the single molecule may refer to, for example, one type of molecule having a plurality of functions, and may be, for example, one nucleic acid (e.g., a DNA or an RNA) including a nucleic acid part formed as the linker, a nucleic acid part formed as the barcode sequence, and a part formed as the target substance capturing part.
  • one nucleic acid e.g., a DNA or an RNA
  • the complex molecule may be, for example, a molecular assembly including two or more kinds of molecules (e.g., a conjugate of two or more kinds of molecules), and may be, for example, a conjugate of nucleic acids including a nucleic acid part formed as the linker and a nucleic acid part formed as the barcode sequence and polypeptides (e.g., proteins or parts thereof, or oligopeptides) formed as the target substance capturing part.
  • nucleic acids including a nucleic acid part formed as the linker and a nucleic acid part formed as the barcode sequence and polypeptides (e.g., proteins or parts thereof, or oligopeptides) formed as the target substance capturing part.
  • the molecule 100 illustrated in FIG. 3 includes a linker 1 , a collection sequence part 2 , an amplification sequence part 3 , a barcode sequence part 4 , a unique molecular identifier (UMI) part 5 , a target substance capturing part 6 , and a bioparticle capturing part 7 .
  • the molecule 100 is immobilized on the surface 101 via the linker 1 .
  • the collection sequence part 2 , the amplification sequence part 3 , the barcode sequence part 4 , and the UMI part 5 may be formed as a series of nucleic acids (particularly DNAs).
  • the target substance capturing part 6 is a nucleic acid
  • the target substance capturing part 6 may also be formed together with the collection sequence part 2 , the amplification sequence part 3 , the barcode sequence part 4 , and the UMI part 5 as a series of nucleic acids (particularly DNAs).
  • one end close to the immobilized portion between the surface 101 and the molecule 100 may be a 5′ end, and the other end may be a 3′ end.
  • the linker 1 may be a linker cleavable by stimulation, and is, for example, a linker cleavable by photic stimulation or chemical stimulation.
  • the photic stimulation is suitable particularly for selectively stimulating a specific position in the cleavage step to be described later.
  • the linker 1 may include any one selected from an arylcarbonylmethyl group, a nitroaryl group, a coumarin-4-ylmethyl group, an arylmethyl group, a metal containing group, and other groups as a linker cleavable by photic stimulation.
  • a group what is described in, for example, Photoremovable Protecting Groups in Chemistry and Biology: Reaction Mechanisms and Efficacy, Chem. Rev. 2013, 113, 119-191 may be used.
  • the arylcarbonylmethyl group may be a phenacyl group, an o-alkylphenacyl group, or a p-hydroxyphenacyl group.
  • the nitroaryl group may be, for example, an o-nitrobenzyl group, an o-nitro-2-phenethyloxycarbonyl group, or o-nitroanilide.
  • the arylmethyl group may be, for example, one into which a hydroxy group is introduced, or one into which no hydroxy group is introduced.
  • the linker 1 is a linker cleavable by photic stimulation
  • the linker may preferably be cleaved by light having a wavelength of 360 nm or more.
  • the linker may be a linker that is preferably cleaved at an energy of 0.5 ⁇ J/ ⁇ m 2 or less. (Light-sheet fluorescence microscopy for quantitative biology, Nat Methods. 2015 January; 12(1): 23-6. doi: 10.1038/nmeth.3219).
  • a linker that is cleaved by light having the wavelength described above or at the energy described above it is possible to reduce cell damage (particularly, cleavage of DNA or RNA) that may occur when photic stimulation is applied.
  • the linker may be a linker cleaved by light in a short wavelength region, particularly light in a wavelength region of 360 nm to 410 nm, or may be a linker cleaved by light in a near infrared region or an infrared region, particularly light in a wavelength region of 800 nm or more.
  • the linker is preferably a linker cleaved by the light in the short wavelength region described above or the light in the near infrared region or the infrared region described above.
  • the linker 1 may include, for example, a disulfide bond or a restriction endonuclease recognition sequence as a linker cleavable by chemical stimulation.
  • a reducing agent such as tris(2-carboxyethyl)phosphine (TCEP), dithiothreitol (DTT), or 2-mercaptoethanol is used.
  • TCEP tris(2-carboxyethyl)phosphine
  • DTT dithiothreitol
  • 2-mercaptoethanol 2-mercaptoethanol
  • restriction endonuclease activity is an amount of endonuclease for completely degrading 1 ⁇ g of ADNA for one hour at 37° C. in principle in 50 ⁇ l of each endonuclease reaction solution, and the amount of the endonuclease is adjusted according to an amount of the restriction endonuclease recognition sequence.
  • the molecule 100 may include a plurality of cleavable linkers.
  • the plurality of linkers may be connected to one another in series.
  • the collection sequence part 2 includes a nucleic acid used for collecting a molecule 100 released from a bioparticle when the bioparticle is disrupted in the disruption step to be described later.
  • the nucleic acid may be a DNA or an RNA, and is particularly a DNA.
  • a bead on which a nucleic acid complementary to the nucleic acid is immobilized may be used. Such a bead makes it possible to efficiently collect the molecule 100 having the collection sequence part 2 .
  • a base sequence of the nucleic acid included in the collection sequence part 2 may be appropriately set by a person skilled in the art.
  • the amplification sequence part 3 may include, for example, a nucleic acid having a primer sequence used for amplifying a nucleic acid or a promoter sequence used for transcribing a nucleic acid in the analysis step to be described later.
  • the nucleic acid may be a DNA or an RNA, and is particularly a DNA.
  • the amplification sequence part 3 may have both a primer sequence and a promoter sequence.
  • the primer sequence may be, for example, a PCR handle.
  • the promoter sequence may be, for example, a T7 promoter sequence.
  • the barcode sequence part 4 includes a nucleic acid having a barcode sequence.
  • the nucleic acid may be particularly a DNA or an RNA, and more particularly a DNA.
  • the barcode sequence may be used, for example, to identify a captured bioparticle (particularly a cell or an exosome), and particularly, may be used as an identifier for distinguishing a bioparticle isolated into one microspace from a bioparticle isolated into another microspace.
  • the barcode sequence may be used as an identifier for distinguishing a target capturing molecule including one barcode sequence from a target capturing molecule including another barcode sequence.
  • the barcode sequence may be associated with a bioparticle to which the target capturing molecule including the barcode sequence is bound.
  • the barcode sequence may be associated with a microspace in which the bioparticle to which the target capturing molecule including the barcode sequence is bound is isolated, and furthermore, may be associated with information regarding a position of the microspace (hereinafter also referred to as “position information”).
  • the position information may be for identifying a position on the surface 101 , and is, for example, information regarding XY coordinates, but is not limited thereto.
  • An ID number may be assigned to the barcode sequence associated with the position information. The ID number may be used in the steps subsequent to the cleavage step. The ID number may correspond to the barcode sequence on a one-to-one basis, and may be used as data corresponding to the barcode sequence in the steps subsequent to the cleavage step.
  • a plurality of target capturing molecules immobilized within a certain region of the surface 101 may have the same barcode sequence.
  • the certain region and the barcode sequence are associated with each other.
  • the target capturing molecule including the barcode sequence can be associated with a position at which one bioparticle is present.
  • a region R in which a plurality of target capturing molecules 100 including the same barcode sequence is immobilized may be smaller than a size of a bioparticle (denoted as a cell in FIG. 2 ).
  • the surface used in the bioparticle analysis method according to the present technology may have a plurality of regions in each of which a plurality of target capturing molecules having the same barcode sequence is immobilized.
  • the barcode sequence may be different for each region.
  • a size of each region e.g., a diameter, a long diameter, a length of a long side, or the like as a maximum dimension of the region
  • the plurality of regions may be arranged at an interval, for example, such that a bioparticle captured by a target capturing molecule immobilized in one region is not captured by a target capturing molecule immobilized in another region.
  • the interval may be, for example, a distance equal to or larger than the size of the bioparticle, and may be preferably a distance larger than the size of the bioparticle.
  • the number of a plurality of regions is preferably larger than the number of bioparticles applied to the surface 101 in the capture step. This prevents two bioparticles from being captured in one region.
  • target capturing molecules whose sequences include a known barcode sequence may be immobilized in a predetermined region.
  • the surface 101 has a plurality of regions, and a plurality of target capturing molecules immobilized in each of the plurality of regions may include the same barcode sequence.
  • the plurality of regions may be set to be smaller than a size of a bioparticle to be captured.
  • the surface 101 configured as described above makes it possible to associate each of the plurality of regions with a barcode sequence included in the plurality of target capturing molecules immobilized in each of the regions.
  • a region in which target capturing molecules including the same barcode sequence are immobilized as described above is also referred to as a spot in the present specification. That is, a size of the spot may be, for example, 50 ⁇ m or less, preferably 10 ⁇ m or less, and more preferably 5 ⁇ m or less.
  • the surface 101 configured as described above makes it possible to associate a barcode sequence included in a certain target capturing molecule with a position at which the certain target capturing molecule is present, at a time point when the target capturing molecules are immobilized on the surface 101 .
  • immobilization for example, biotin is bound to the linker 1 of the target capturing molecule, and streptavidin is bound to the surface 101 on which the target capturing molecule is immobilized, such that the biotin and the streptavidin are bound to each other, thereby immobilizing the target capturing molecule on the surface 101 .
  • target capturing molecules each including a barcode sequence may be randomly arranged on the surface 101 .
  • a barcode sequence included in a certain target capturing molecule is associated with a position at which the certain target capturing molecule is present by reading the barcode sequence included in the immobilized target capturing molecule.
  • the reading can be performed by a method such as sequencing by synthesis, sequencing by ligation, or sequencing by hybridization.
  • a barcode sequence included in a certain target capturing molecule may not be associated with a position at which the certain target capturing molecule is present. Since a bioparticle and a target capturing molecule are isolated from each other into a microspace by the isolation step to be described later, the bioparticle and the target capturing molecule (particularly a barcode sequence included in the target capturing molecule) are associated with each other on a one-to-one basis.
  • a bead e.g., gel bead
  • the bead e.g., gel bead
  • a size of the bead may be, for example, 50 ⁇ m or less, preferably 10 ⁇ m or less, and more preferably 5 ⁇ m or less.
  • a combination of biotin and streptavidin may be used.
  • biotin is bound to the linker 1 of the target capturing molecule
  • streptavidin is bound to the bead, such that the biotin and the streptavidin are bound to each other, thereby immobilizing the target capturing molecule on the bead.
  • the surface 101 may have a plurality of recesses therein.
  • one spot or one bead may be arranged in each of the plurality of recesses.
  • the spots or the beads can be more easily arranged on the surface 101 through the plurality of recesses.
  • the recess preferably has a size in which one bead is placed.
  • the recess may have a circular shape, an elliptical shape, a hexagonal shape, or a quadrangular shape, but the shape of the recess is not limited thereto.
  • some portions of the surface 101 may be in a different state from the other portions of the surface 101 .
  • some surface portions where the spots or the beads are arranged may be hydrophilic, and the other surface portions may be hydrophobic, or the other surface portions may be hydrophobic while each having a protrusion.
  • a method for imparting hydrophilicity to the surface reactive ions may be etched in the presence of oxygen, or deep ultraviolet light may be irradiated in the presence of ozone. In such a method, a mask may be used with through holes formed in the portions to which hydrophilicity is to be imparted.
  • a spray-on-silicone for example, Techspray 2101-12S
  • a mask may also be used with through holes formed in the portions to which hydrophobicity is to be imparted.
  • the target capturing molecule can also be synthesized on the substrate using a DNA microarray preparation technique or the like.
  • a target capturing molecule can be synthesized at a specific position using a technology such as a digital micromirror device (DMD), a liquid crystal shutter, or a spatial light phase modulator, which is used for photolithography.
  • DMD digital micromirror device
  • a liquid crystal shutter or a spatial light phase modulator, which is used for photolithography.
  • a method for the synthesis is described, for example, in Basic Concepts of Microarrays and Potential Applications in Clinical Microbiology, CLINICAL MICROBIOLOGY REVIEWS, October 2009, p. 611-633.
  • a target capturing molecule is synthesized on the substrate by the above-described synthesis method
  • information on a position at which the target capturing molecule is synthesized is acquired when the target capturing molecule is synthesized, and a barcode sequence is associated with the position information.
  • an ID number may be assigned.
  • all of the target capturing molecules immobilized on the surface may include a common oligo sequence.
  • a nucleic acid that is fluorescently labeled while having a sequence complementary to the oligo sequence it is possible to confirm a position at which a target capturing molecule is immobilized (particularly a position of a spot or a position of a bead), particularly in a dark field.
  • the fluorescent label makes it easy to grasp the position at which the target capturing molecule is immobilized.
  • the UMI part 5 may include a nucleic acid, particularly a DNA or an RNA, and more particularly a DNA.
  • the UMI part 5 may have a sequence of, for example, 5 to 30 bases, particularly 6 to 20 bases, and more particularly 7 to 15 bases.
  • the UMI part 5 may be formed to have a different sequence between target capturing molecules immobilized on the surface 101 .
  • the number of types of UMI sequences is 4 to the power of 10, that is, one million or more.
  • the UMI part 5 may be used to quantify a target substance.
  • a UMI sequence may be added to a cDNA obtained by reversely transcribing the mRNA as the target substance, for example, in the analysis step to be described later.
  • a large number of cDNAs obtained by amplifying a cDNA obtained by reverse transcription from one mRNA molecule have the same UMI sequence, but a large number of cDNAs obtained by amplifying a cDNA obtained by transcription from another mRNA molecule having the same sequence as the one mRNA molecule have a different UMI sequence from those from the one mRNA molecule. Therefore, the number of copies of mRNA can be determined by counting the number of types of UMI sequences having the same cDNA sequence.
  • the UMI part 5 may be formed to have a different sequence between a plurality of target capturing molecules including the same barcode sequence while being immobilized in one region R (e.g., the spot or the bead). That is, the plurality of target capturing molecules immobilized in the region R (e.g., the spot or the bead) may have different UMIs while having the same barcode sequence.
  • the target substance capturing part 6 includes a component for capturing a molecule contained in a bioparticle.
  • the component may be, for example, a nucleic acid or a protein.
  • the nucleic acid may have, for example, a poly T sequence in order to comprehensively capture mRNAs contained in a cell.
  • the nucleic acid may have a sequence complementary to the target sequence.
  • the component is a protein
  • the protein may be, for example, an antibody.
  • the component may be an aptamer or a molecular imprinted polymer.
  • the target substance capturing part 6 may include two or more kinds of components for capturing a molecule contained in a cell.
  • the target substance capturing part 6 may include both a protein and a nucleic acid, and may include, for example, both an antibody and a poly T sequence. This makes it possible to simultaneously detect both a protein and an mRNA.
  • the bioparticle capturing part 7 includes a component for capturing a bioparticle, and particularly includes a component for capturing a cell.
  • the component may be, for example, an antibody, an aptamer, or an oleyl group.
  • the antibody may be an antibody that is bound to a constituent (particularly a surface antigen) present on a surface of a bioparticle such as a cell or an exosome.
  • the aptamer may be a nucleic acid aptamer or a peptide aptamer.
  • the aptamer may also be bound to a constituent (particularly a surface antigen) present on a surface of a bioparticle such as a cell or an exosome.
  • the oleyl group may bind a bioparticle including a lipid bilayer membrane such as a cell or an exosome thereto.
  • the surface 101 may preferably be a surface of a transparent substrate.
  • the substrate may be entirely transparent, or may be transparent only in a portion on which a target capturing molecule is immobilized.
  • the surface of the substrate is preferably planar to bring a specimen in contact therewith in a good condition.
  • the transparent substrate may be, for example, a glass substrate or a resin substrate.
  • the substrate may be, for example, a slide glass. The transparency makes it easy to select a bioparticle to be cleaved in the detection step and the cleavage step to be described later.
  • the number and density of molecules 100 bound to the surface 101 can be increased, for example, by increasing a surface area of surface 101 .
  • the plurality of molecules 100 may be connected to each other in series.
  • a cleavage condition between the substrate and a molecule 100 is preferably different from a cleavage condition between two molecules 100 .
  • the cleaved molecule may be bound to another adjacent bioparticle.
  • the different cleavage conditions make it possible to prevent the cleaved molecule from being bound to another adjacent bioparticle.
  • a linker binding a molecule 100 to the substrate may be a linker cleavable by photic stimulation, and a linker binding a molecule 100 to another molecule 100 may be a linker cleavable by chemical stimulation.
  • a linker binding a molecule 100 to the substrate may be a linker cleavable by chemical stimulation, and a linker binding a molecule 100 to another molecule 100 may be a linker cleavable by another type of chemical stimulation.
  • the former may include a restriction endonuclease recognition sequence, and the latter may include another restriction endonuclease recognition sequence.
  • the former may include a disulfide bond
  • the latter may include a restriction endonuclease recognition sequence.
  • molecules may be bound to each other by an amino acid, and may be cleaved from each other in the disruption step to be described later (particularly simultaneously with cytolysis) by a reagent (e.g., proteinase K) used for the cytolysis.
  • a reagent e.g., proteinase K
  • a bioparticle (a cell in FIG. 2 B ) is captured by a molecule 100 .
  • the bioparticle is captured by a bioparticle capturing part 7 of the molecule 100 .
  • the bioparticle and the bioparticle capturing part 7 may be bound to each other in a specific or non-specific manner.
  • a surface antigen of the cell may be bound to an antibody or aptamer contained in the bioparticle capturing part 7 , such that the cell is captured by the molecule 100 .
  • the antibody and the aptamer may be specific or non-specific.
  • a lipid bilayer membrane of the cell may be bound to an oleyl group contained in the bioparticle capturing part 7 , such that the cell is captured by the molecule 100 .
  • a surface constituent (a component constituting a lipid bilayer membrane) of the bioparticle may be bound to an oleyl group contained in the bioparticle capturing part 7 , such that the bioparticle is captured by the molecule 100 .
  • the surface constituent of the exosome may be bound to an antibody or an aptamer contained in the bioparticle capturing part 7 , such that the bioparticle is captured by the molecule 100 .
  • the capture step S 102 may include an application step of applying a bioparticle to the surface 101 .
  • the application step may be performed, for example, in such a manner that a bioparticle-containing sample (e.g., a bioparticle-containing liquid) is brought into contact with the surface 101 .
  • a bioparticle-containing sample e.g., a bioparticle-containing liquid
  • the bioparticle-containing sample may be dropped onto the surface 101 .
  • a plurality of molecules bound to one bioparticle may have the same barcode sequence. This makes it possible to associate one barcode sequence with one bioparticle. Furthermore, UMI parts included in the plurality of molecules preferably have different sequences. This makes it possible to determine the number of copies of, for example, mRNA.
  • a label may be assigned to the bioparticle.
  • the label may be used to select a bioparticle to be released from the surface, for example, in the cleavage step to be described later.
  • the label assigned to the bioparticle is preferably an antibody having a label.
  • an antibody labeled with a fluorochrome hereinafter, also referred to as a “fluorochrome-labeled antibody” may be bound to the bioparticle.
  • An example of such an antibody is illustrated in FIG. 2 B .
  • an antibody 10 bound to (particularly specifically bound to) a surface antigen 12 of a bioparticle may be bound to the antigen.
  • the antibody 10 may be labeled with, for example, a fluorochrome 11 .
  • a bioparticle to be cleaved can be selected on the basis of fluorescence generated from the fluorochrome that labels the antibody, for example, in the detection step to be described later.
  • the fluorescence can also be used to determine whether to isolate a bioparticle into a microspace, for example, in the determination step to be described later.
  • the fluorochrome-labeled antibody may be bound to the bioparticle before the bioparticle is captured on the surface 101 , or may be bound to the bioparticle after the bioparticle is captured on the surface 101 .
  • a label may be assigned to a molecule 100 .
  • the label may be used to select a bioparticle to be released from the surface, for example, in the cleavage step to be described later.
  • the label assigned to the molecule 100 is, for example, a fluorochrome.
  • some of the nucleic acids constituting the molecule 100 may be nucleic acids labeled with a fluorochrome.
  • an antibody to which a nucleic acid including an antibody barcode sequence is bound may be bound to a bioparticle.
  • the antibody barcode sequence is a barcode sequence for identifying a nucleic acid-bound antibody.
  • a nucleic acid-bound antibody illustrated in FIG. 7 is bound to the bioparticle instead of or in addition to the fluorochrome-labeled antibody described with reference to FIG. 2 B .
  • the nucleic acid-bound antibody illustrated in FIG. 7 includes an antibody 10 and nucleic acids bound to the antibody.
  • the nucleic acids include a first nucleic acid 21 , a second nucleic acid 22 , and a third nucleic acid 23 . These nucleic acids may be arranged in the order illustrated in FIG. 7 , or may be arranged in another order.
  • the first nucleic acid 21 may include an amplification primer sequence. Since the first nucleic acid 21 includes an amplification primer sequence, the barcode sequence part 4 , the unique molecular identifier (UMI) part 5 , and/or the like included in the molecule 100 can be assigned to the second nucleic acid 22 and the third nucleic acid 23 to be described later at the time of amplification. Furthermore, a sequence for sequence processing, e.g., an adapter sequence, or the like can be assigned.
  • UMI unique molecular identifier
  • the second nucleic acid 22 may include an antibody barcode sequence.
  • the antibody barcode sequence may be used to distinguish a nucleic acid-bound antibody bound to one bioparticle from a nucleic acid-bound antibody bound to another bioparticle.
  • the antibody barcode sequence may be different for each type of antibody, or the antibody barcode sequence may be different for each type of bioparticle. This makes it possible to identify a bioparticle to which a nucleic acid-bound antibody was bound after the bioparticle is disrupted in the disruption step to be described later.
  • the third nucleic acid 23 may include a poly A sequence.
  • the nucleic acids including the first nucleic acid 21 and the second nucleic acid 22 as described above can be captured by the poly T sequence included in the target substance capturing part 6 of the molecule 100 via the third nucleic acid 23 .
  • a complex of the nucleic acids and the molecule 100 is formed through the capture step.
  • amplifying the complex for example, using the first nucleic acid 21 , a nucleic acid to which an antibody barcode sequence is assigned as a second nucleic acid 22 is generated in the molecule 100 .
  • the nucleic acid generated by the amplification has an antibody barcode sequence, and the antibody barcode sequence is different, for example, for each type of antibody as described above, that is, associated with the type of antibody. Therefore, information regarding the type and/or the number of nucleic acid-bound antibodies is maintained in the form of an antibody barcode sequence in a product of the amplification, and the type and/or the number of nucleic acid-bound antibodies associated with the antibody barcode sequence can be identified, for example, from the sequence and/or the number of nucleic acids having the antibody barcode sequence. This makes it possible to identify the type and the number of nucleic acid-bound antibodies bound to a bioparticle (i.e., a cell or the like). The identification may be performed, for example, in the analysis step to be described later. The sequence analysis of the amplification product for the identification may be performed by, for example, NGS.
  • the capture step S 102 may include an incubation step for binding the bioparticle and the bioparticle capturing part to each other. Incubation conditions such as incubation time and temperature may be determined depending on the type of the bioparticle capturing part to be used.
  • a removal step of removing a bioparticle that has not bound to the molecule 100 may be performed. Furthermore, after the capture step S 102 , a removal step of removing a substance unnecessary in the cleavage step, such as an antibody that has not bound to the bioparticle, may be performed.
  • the removal step may include washing the surface 101 using a liquid, e.g., a buffer.
  • the linker 1 is cleaved, and the bioparticle captured in the capture step S 102 is released from the surface 101 .
  • the captured state of the bioparticle is maintained by the bioparticle capturing part 7 .
  • the captured state may be maintained until the bioparticle is completely isolated in the isolation step S 104 to be described later, and may be maintained, for example, until the bioparticle is completely disrupted in the disruption step S 105 to be described later.
  • the cleavage may be performed on an entire portion of the surface 101 , or may be performed on a partial portion of the surface 101 .
  • the partial portion of the surface may be selected, for example, on the basis of a detection result of the detection step to be described below.
  • the cleavage may be performed to release all of the bioparticles captured on the surface 101 from the surface 101 , or may be performed to release some of the bioparticles captured on the surface 101 from the surface 101 . In the latter case, some of the bioparticles may be selected, for example, on the basis of a detection result of the detection step to be described below.
  • the bioparticle to be released from the surface 101 may be selected on the basis of a label of the bioparticle or a label of the molecule 100 .
  • the label of the bioparticle may be, for example, a fluorochrome constituting the fluorochrome-labeled antibody described in section (3-2) above, or a label (particularly a fluorochrome) present inside the bioparticle.
  • the label of the molecule 100 may be, for example, a label assigned to the molecule 100 described in section (3-2) above, particularly a fluorochrome.
  • the cleavage step may be performed without performing the detection step after the capture step.
  • the cleavage step S 103 may include a detection step of detecting light generated from a bioparticle or light from a substance bound to a bioparticle, and a linker cleavage step of cleaving a linker on the basis of a detection result in the detection step to release the bioparticle from the surface 101 .
  • the linker cleavage step may be performed without performing the detection step. By omitting the detection step, the number of steps in the analysis method according to the present technology can be reduced.
  • the cleavage step S 103 may include a detection step of detecting any one or two or more of light (e.g., scattered light and/or autofluorescence) derived from a bioparticle, light (e.g., fluorescence) derived from a target capturing molecule, light (e.g., fluorescence) derived from an antibody bound to a bioparticle, a form of a bioparticle (e.g., morphology (an image acquired in bright field, in phase difference, or in dark field and a form characterized by image processing, particularly a form acquired by morphology processing), a state in which two or more bioparticles (cells or the like) are bound to each other, or the like), and a feature of a bioparticle predicted from morphological information about the bioparticle (e.g., a cell type or a cell state (living cell or dead cell)).
  • light e.g., scattered light and/or autofluorescence
  • light e.g., fluor
  • the light, form, feature, and the like may be detected, for example, by an observation device including an objective lens, particularly a microscope device.
  • the light, form, and feature may be detected, for example, by an imaging element or by a photodetector.
  • a target capturing molecule to be cleaved in the linker cleavage step to be described later may be selected, or a bioparticle to be released from the surface 101 in the cleavage step S 104 may be selected.
  • the imaging element may acquire an image of the surface 101 or an image of bioparticles captured on the surface 101 , and a bioparticle to be released may be selected on the basis of the acquired image.
  • the cleavage step S 103 includes a linker cleavage step of cleaving the linker 1 .
  • a linker cleavage step of cleaving the linker 1 By cleaving the linker 1 , a bioparticle to which the target capturing molecule is bound is released from the surface 101 .
  • the molecule 100 is released from the surface 101 , and accordingly, the bioparticle is also released from the surface 101 .
  • the linker may be cleaved, for example, by stimulation such as chemical stimulation or photic stimulation.
  • the photic stimulation is particularly suitable for selectively stimulating a specific narrow range.
  • the stimulation in the cleavage step S 103 may be provided by a stimulation application device.
  • the driving of the stimulation application device may be controlled, for example, by an information processing device such as a general-purpose computer.
  • the information processing device may drive the stimulation application device to selectively apply stimulation to a position of a bioparticle to be released.
  • An example of the stimulation application device that may be adopted will be described below.
  • a light irradiation device may be used as the stimulation application device.
  • the light irradiation device may be, for example, a digital micromirror device (DMD) or a liquid crystal display device.
  • a selected position on the surface 101 can be irradiated with light by a micromirror constituting the DMD.
  • the liquid crystal display device may be, for example, a reflective liquid crystal display, and a specific example thereof may include SXRD (Sony Corporation). By controlling a liquid crystal of the liquid crystal display device, a selected position on the surface 101 can be irradiated with light.
  • a liquid crystal shutter or a spatial light modulator may be used to selectively apply photic stimulation to a position of a cell.
  • the liquid crystal shutter or the spatial light modulator can also apply the photic stimulation to a selected position.
  • a wavelength of light to be irradiated may be appropriately selected by a person skilled in the art depending on what type of linker the target capturing molecule includes.
  • the chemical stimulation may be applied by bringing a reagent for cleaving the linker 1 into contact with the surface 101 .
  • the reagent may be determined depending on the type of linker 1 .
  • the reagent may be a reducing agent capable of cleaving the bond, and examples thereof may include tris(2-carboxyethyl)phosphine (TCEP), dithiothreitol (DTT), and 2-mercaptoethanol.
  • TCEP tris(2-carboxyethyl)phosphine
  • DTT dithiothreitol
  • 2-mercaptoethanol 2-mercaptoethanol
  • the reagent may be a restriction endonuclease corresponding to each restriction endonuclease recognition sequence.
  • 1 U of restriction endonuclease activity is an amount of endonuclease for completely degrading 1 ⁇ g of ⁇ DNA for one hour at 37° C. in principle in 50 ⁇ l of each endonuclease reaction solution, and the amount of the endonuclease may be adjusted according to an amount of the restriction endonuclease recognition sequence.
  • At least one bioparticle released by the cleavage in the cleavage step S 103 may be collected, for example, in a liquid such as a buffer.
  • the liquid may be, for example, a hydrophilic liquid.
  • the bioparticle-containing liquid obtained by the collection may be used in the isolation step S 104 to be described later.
  • a fluid force caused when the liquid such as a buffer flows may be used, the bioparticle may be floated in the liquid under vibration, or the bioparticle may be floated in the liquid using the gravity or the like.
  • the vibration may be, for example, vibration of the analysis substrate 102 or vibration of the liquid in which the bioparticle is contained.
  • the analysis substrate 102 may be moved such that the surface 101 faces the direction of gravity.
  • the bioparticle released from the surface 101 in the cleavage step is isolated in a microspace.
  • the molecule 100 can be bound to a target substance included, for example, in the bioparticle. This makes it possible to associate the target substance, for example, with the barcode sequence included in the molecule 100 . Using the barcode sequence information, it is possible to analyze a target substance, particularly a single cell.
  • the microspace may be, for example, a space in an emulsion particle or a space in a well.
  • one bioparticle (particularly one bioparticle to which at least one molecule 100 is bound) is isolated in one emulsion particle or one well.
  • the isolation step S 104 may include a determination step of determining whether to isolate a bioparticle into a microspace, and a particle isolation step of isolating the bioparticle determined to be isolated into the microspace in the determination step. This makes it possible to isolate only intended bioparticles. Therefore, for example, unintended bioparticles can be excluded from targets in the analysis step to be described later, thereby improving analysis efficiency.
  • the determination may be performed, for example, on the basis of light generated from a bioparticle (e.g., scattered light and/or autofluorescence), light generated from a substance bound to a bioparticle, or a morphological image.
  • the substance bound to the bioparticle may be, for example, a target capturing molecule, or may be an antibody (particularly a fluorochrome-labeled antibody) bound to the bioparticle.
  • the scattered light generated from the bioparticle may be, for example, forward scattered light and/or side scattered light.
  • a doublet can be detected from a height and/or an area value of a signal acquired by detecting the scattered light.
  • a single cell can be determined based on morphological image information.
  • the bioparticle is a dead cell can be determined from scattered light and/or a morphological image or fluorescence after the dead cell is dyed with a dead cell dyeing reagent, thereby removing the dead cell.
  • the determination step may be performed immediately before the isolation step, thereby making it possible to reliably isolate only a single cell to which the barcode is assigned.
  • the particle isolation step may be performed without performing the determination step. By omitting the determination step, the number of steps in the analysis method according to the present technology can be reduced.
  • the determination step may be performed in the detection step described in section (3-3-1) above or the linker cleavage step described in section (3-3-2) above, rather than being performed in the isolation step.
  • a bioparticle or a bioparticle population selected as a result of the determination in the detection step or the linker cleavage step is subjected to the particle isolation step to be described later. In this case, it is not necessary to use a device such as a cell sorter.
  • the determination step it is determined whether a bioparticle released in the step S 104 is to be isolated into a microspace. As described above, the determination may be performed on the basis of light generated from the bioparticle or light generated from the substance bound to the bioparticle.
  • the determination step may include, for example, an irradiation step of irradiating the bioparticle with light and a detection step of detecting light generated by the irradiation.
  • the irradiation step may be performed, for example, by a light irradiation unit that irradiates the bioparticle with light.
  • the light irradiation unit may include, for example, a light source that emits light.
  • the light irradiation unit may include an objective lens that condenses light on the bioparticle.
  • the light source may be appropriately selected by a person skilled in the art according to the purpose of analysis, and examples thereof may include a laser diode, an SHG laser, a solid-state laser, a gas laser, a high-luminance LED, a halogen lamp, and a combination of two or more thereof.
  • the light irradiation unit may include another optical element, if necessary, in addition to the light source and the objective lens.
  • the detection step may be performed, for example, by a detection unit that detects light generated from a bioparticle or a substance bound to the bioparticle.
  • the detection unit may detect light from the bioparticle or the substance bound to the bioparticle generated by the light irradiation unit irradiating light, e.g., scattered light and/or fluorescence.
  • the detection unit may include, for example, a condenser lens that condenses light generated from the bioparticle and a detector.
  • a PMT, a photodiode, a CCD, a CMOS, or the like may be used, but the detector is not limited thereto.
  • the detection unit may include another optical element, if necessary, in addition to the condenser lens and the detector.
  • the detection unit may further include a spectroscopic unit.
  • optical components constituting the spectroscopic unit may include a grating, a prism, and an optical filter.
  • the spectroscopic unit can detect, for example, light having a wavelength to be detected distinguishably from light having another wavelength.
  • the detection unit may convert the detected light into an analog electric signal by photoelectric conversion.
  • the detection unit may further convert the analog electric signal into a digital electric signal by AD conversion.
  • the determination step may be performed by a determination unit that performs determination processing as to whether to determine a bioparticle on the basis of the light detected in the detection step.
  • the processing performed by the determination unit may be realized by, for example, an information processing device such as a general-purpose computer, particularly a processing unit included in the information processing device.
  • the isolation step S 104 includes a particle isolation step of isolating a bioparticle into a microspace.
  • the microspace may refer to a space having a dimension capable of accommodating one bioparticle to be analyzed.
  • the dimension may be appropriately determined according to a factor such as a size of the bioparticle.
  • the microspace may have a dimension capable of accommodating two or more bioparticles to be analyzed. In this case, however, not only one bioparticle but also two or more bioparticles may be accommodated in one microspace.
  • the bioparticles in the microspace in which two or more bioparticles are accommodated may be excluded from targets to be disrupted in the disruption step to be described layer, or may be excluded from targets to be analyzed in the analysis step to be described later.
  • a complex of a target capturing molecule and a target substance may be generated in the disruption step to be described later.
  • a plurality of microspaces used in the present technology is preferably separated from each other so that the complex generated in one microspace does not shift to another microspace.
  • the microspaces separated as described above can include spaces in wells and spaces in emulsion particles. That is, in a preferred embodiment of the present technology, the microspaces may be spaces in wells and spaces in emulsion particles.
  • an example of the particle isolation step in a case where the microspace is a space in a well and an example of the particle isolation step in a case where the microspace is a space in an emulsion particle will be individually described.
  • FIG. 4 A schematic diagram of an example of a well used to perform the particle isolation step is illustrated in FIG. 4 .
  • a plurality of wells 40 for example, each having a dimension capable of accommodating one bioparticle, may be formed in a surface of a substrate 41 .
  • a bioparticle 43 is isolated into a space in the well 40 as illustrated in FIG. 4 .
  • one bioparticle may enter a space in one well, such that the bioparticle is isolated into the microspace.
  • the particle isolation step may be performed without performing the determination step described in section (3-4-1) above.
  • a device for putting one bioparticle in one well such as a cell sorter or a single cell dispenser
  • a substrate e.g., a plate
  • a substrate e.g., a plate
  • a commercially available device may be used.
  • the device may include, for example, a light irradiation unit that irradiates the bioparticles with light, a detection unit that detects light from a bioparticle, a determination unit that determines whether to put the bioparticle into a well on the basis of the detected light, and a distribution unit that distributes into the well the bioparticle determined to be put into the well.
  • a light irradiation unit that irradiates the bioparticles with light
  • a detection unit that detects light from a bioparticle
  • a determination unit that determines whether to put the bioparticle into a well on the basis of the detected light
  • a distribution unit that distributes into the well the bioparticle determined to be put into the well.
  • the light irradiation unit and the detection unit perform the detection step, and the determination unit performs the determination step.
  • the distribution unit includes, for example, a microfluidic chip having a nozzle that forms a droplet containing a bioparticle.
  • the device operates a position of the microfluidic chip according to a result of determination by the determination unit to put a droplet containing one bioparticle into a predetermined well.
  • the device controls a direction in which a bioparticle-containing droplet discharged from the nozzle is headed, using a charge applied to the droplet according to a result of determination by the determination unit. This control makes it possible to put a droplet containing one bioparticle into a predetermined well. In this way, one bioparticle is distributed into one well.
  • a bioparticle-containing droplet is discharged from the nozzle 52 provided in the microfluidic chip of the device.
  • the bioparticle contained in the droplet is irradiated with light (e.g., laser light L) by the light irradiation unit 54 , and then, a detection step is performed by the detection unit 55 and light (fluorescence F) is detected.
  • the determination unit (not illustrated) performs a determination step on the basis of the detected light.
  • the distribution unit controls a direction in which the droplet is headed using a charge applied to the droplet. This control makes it possible to collect a droplet containing an intended bioparticle in a predetermined well. Therefore, one bioparticle is distributed into one well.
  • the determination step it is possible to identify, for example, a cell population to which bioparticles according to a detection signal belong, a bioparticle to which a barcode is assigned, or a droplet containing a singlet bioparticle. This makes it possible to collect only droplets containing intended bioparticles. As a result, it is not necessary to exclude data in the analysis step to be described later, thereby improving analysis efficiency.
  • the number of wells provided in one substrate (plate) may be, for example, 1 to 1000, particularly 10 to 800, and more particularly 30 to 500, but the number of wells may be appropriately selected by a person skilled in the art.
  • the emulsion particles may be produced, for example, using a microchannel.
  • the device includes, for example, a channel through which a first liquid forming a dispersoid of an emulsion flows and a channel through which a second liquid forming a dispersion medium flows.
  • the first liquid may contain bioparticles.
  • the device further includes a region where the two liquids contact each other to form an emulsion. An example of the microchannel will be described below with reference to FIG. 6 .
  • the microchannel illustrated in FIG. 6 includes a channel 61 through which a first liquid containing bioparticles flows, and channels 62 - 1 and 62 - 2 through which a second liquid flows.
  • the first liquid forms emulsion particles (dispersoid), and the second liquid forms a dispersion medium of the emulsion.
  • the channel 61 and the channels 62 - 1 and 62 - 2 join together, and the emulsion particles are formed at this junction point.
  • bioparticles 63 are isolated into the emulsion particles.
  • a size of the emulsion particles can be controlled by controlling a flow velocity in these channels.
  • the first liquid and the second liquid are immiscible with each other to form an emulsion.
  • the first liquid may be a hydrophilic liquid and the second liquid may be a hydrophobic liquid, or vice versa.
  • the microchannel illustrated in FIG. 6 may include a channel 64 for introducing a bioparticle-disrupting substance into the emulsion particles.
  • a channel 64 for introducing a bioparticle-disrupting substance into the emulsion particles.
  • the emulsion forming device makes it possible to isolate one bioparticle into one emulsion particle with very high probability, thereby reducing the number of empty emulsion particles. Moreover, the emulsion forming device makes it possible to increase probability that one bioparticle is isolated into one emulsion particle together with one barcode sequence.
  • FIG. 8 A is an example of a microchip used for forming emulsion particles in the device.
  • the microchip 150 illustrated in FIG. 8 A includes a main channel 155 through which bioparticles flow and a collection channel 159 through which particles to be collected among the bioparticles are collected.
  • the microchip 150 includes a particle sorting part 157 .
  • An enlarged view of the particle sorting part 157 is illustrated in FIG. 9 .
  • the particle sorting part 157 includes a connection channel 170 that connects the main channel 155 and the collection channel 159 to each other.
  • a liquid supply channel 161 capable of supplying a liquid to the connection channel 170 is connected to the connection channel 170 .
  • the microchip 150 has a channel structure including the main channel 155 , the collection channel 159 , the connection channel 170 , and the liquid supply channel 161 .
  • FIG. 8 B is a schematic diagram illustrating how emulsion particles are formed and bioparticles are isolated into the formed emulsion particles in the microchip 150 illustrated in FIG. 8 A .
  • the microchip 150 constitutes a part of a bioparticle sorting device 200 including a light irradiation unit 191 , a detection unit 192 , and a control unit 193 in addition to the microchip.
  • the control unit 193 may include a signal processing unit 194 , a determination unit 195 , and a sorting control unit 196 .
  • the bioparticle sorting device 200 is used as the emulsion forming device described above.
  • the determination step S 202 corresponds to the determination step described in section (3-4-1) above.
  • the collection step S 203 corresponds to the particle isolation step described in section (3-4-2) above.
  • the first liquid containing bioparticles flows through the main channel 155 .
  • the first liquid flows from a junction portion 162 toward the particle sorting part 157 in the main channel 155 .
  • the first liquid may be a laminar liquid including a sample liquid containing bioparticles and a sheath liquid, and particularly, may be a laminar liquid in which the periphery of the sample liquid is surrounded by the sheath liquid.
  • a channel structure for forming the laminar flow will be described below.
  • the sheath liquid may contain, for example, a bioparticle-disrupting component, e.g., a cytolytic component.
  • a bioparticle-disrupting component e.g., a cytolytic component.
  • the cytolytic component may be a cytolytic enzyme, e.g., proteinase K.
  • a predetermined temperature e.g., 37° C. to 56° C.
  • the proteinase K is active even at 37° C. or lower, the proteinase K may be incubated, for example, overnight, taking into account that the cytolytic property of the proteinase K decreases in a case where such a lower temperature is employed.
  • the sheath liquid may contain a surfactant (e.g., SDS, Sarkosyl, Tween 20, or Triton X-100). The surfactant makes it possible to enhance the activity of the proteinase K.
  • the sheath liquid may not contain a bioparticle-disrupting component.
  • bioparticles may be physically disrupted.
  • a physical disruption method for example, optical treatment (e.g., optical lysis) or thermal treatment (e.g., thermal lysis) may be adopted.
  • the optical treatment may be performed, for example, by forming plasma or cavitation bubbles in the emulsion particles by irradiating the particles with laser light.
  • the thermal disruption of particles may be achieved by heating the emulsion particles.
  • the microchip 150 has a sample liquid inlet 151 and a sheath liquid inlet 153 . From these inlets, the sample liquid containing bioparticles and the sheath liquid containing no bioparticles are introduced into a sample liquid channel 152 and a sheath liquid channel 154 , respectively.
  • the microchip 150 has a channel structure in which the sample channel 152 through which the sample liquid flows and the sheath liquid channel 154 through which the sheath liquid flows join together at the junction portion 162 to become a main channel 155 .
  • the sample liquid and the sheath liquid join together at the junction portion 162 , for example, to form a laminar flow in which the periphery of the sample liquid is surrounded by the sheath liquid.
  • FIG. 8 B A schematic view of the formation of the laminar flow is illustrated in FIG. 8 B . As illustrated in FIG. 8 B , a laminar flow is formed so that the sample liquid introduced from the sample channel 152 is surrounded by the sheath liquid introduced from the sheath liquid channel 154 .
  • bioparticles are aligned substantially in a line in the laminar flow.
  • the bioparticles P may be aligned substantially in a line in the sample liquid.
  • the channel structure according to the present technology forms a laminar flow including bioparticles flowing substantially in a line.
  • the laminar flow flows through the main channel 155 toward the particle sorting part 157 .
  • the bioparticles flow side by side in the main channel 155 .
  • the determination step S 202 it is determined whether a bioparticle flowing through the main channel 155 is a particle to be collected.
  • the determination may be performed by the determination unit 195 .
  • the determination unit 195 may perform the determination on the basis of light generated by the light irradiation unit 191 irradiating the bioparticle with light. An example of the determination step S 202 will be described in more detail below.
  • the light irradiation unit 191 irradiates a bioparticle flowing through the main channel 155 (particularly the detection region 156 ) in the microchip 150 with light (e.g., excitation light), and the detection unit 192 detects light generated by irradiating the light.
  • the determination unit 195 included in the control unit 193 determines whether the bioparticle is a particle to be collected.
  • the determination unit 195 may perform determination based on scattered light, determination based on fluorescence, or determination based on an image (e.g., one or more of a dark field image, a bright field image, and a phase difference image).
  • the control unit 193 controls a flow in the microchip 150 to collect a particle to be collected into the collection channel 159 .
  • the light irradiation unit 191 irradiates a bioparticle flowing through the channel in the microchip 150 with light (e.g., excitation light).
  • the light irradiation unit 191 may include a light source that emits light and an objective lens that condenses excitation light on a microparticle flowing in the detection region.
  • the light source may be appropriately selected by a person skilled in the art according to the purpose of analysis, and examples thereof may include a laser diode, an SHG laser, a solid-state laser, a gas laser, a high-luminance LED, a halogen lamp, and a combination of two or more thereof.
  • the light irradiation unit may include another optical element, if necessary, in addition to the light source and the objective lens.
  • the detection unit 192 detects scattered light and/or fluorescence generated from a microparticle by the light irradiation unit 191 irradiating light.
  • the detection unit 192 may include a condenser lens that collects fluorescence and/or scattered light generated from the bioparticle and a detector.
  • a PMT, a photodiode, a CCD, a CMOS, or the like may be used, but the detector is not limited thereto.
  • the detection unit 192 may include another optical element, if necessary, in addition to the condenser lens and the detector.
  • the detection unit 192 may further include a spectroscopic unit.
  • optical components constituting the spectroscopic unit may include a grating, a prism, and an optical filter.
  • the spectroscopic unit can detect, for example, light having a wavelength to be detected distinguishably from light having another wavelength.
  • the detection unit 192 may convert the detected light into an analog electric signal by photoelectric conversion.
  • the detection unit 192 may further convert the analog electric signal into a digital electric signal by AD conversion.
  • the signal processing unit 194 included in the control unit 193 may process a waveform of the digital electric signal obtained by the detection unit 192 to generate information (data) regarding a feature of light to be used for determination by the determination unit 105 .
  • the signal processing unit 194 may acquire, for example, one, two, or three of a width of the waveform, a height of the waveform, and an area of the waveform from the waveform of the digital electric signal.
  • the information regarding the feature of the light may include, for example, a time at which the light is detected.
  • the processing performed by the signal processing unit 194 described above may be performed particularly in an embodiment in which the scattered light and/or the fluorescence is detected.
  • the determination unit 195 included in the control unit 193 determines whether the bioparticle is a particle to be collected.
  • a waveform of a digital electric signal obtained by the detection unit 192 is processed by the control unit 193 , and then, the determination unit 195 determines whether the bioparticle is a particle to be collected on the basis of information regarding a feature of light generated by the processing. For example, in a case where determination is made on the basis of scattered light, a feature of the bioparticle concerning its outer shape and/or internal structure may be specified, and it may be determined on the basis of the feature whether or not the bioparticle is a particle to be collected.
  • bioparticle such as a cell
  • a bioparticle such as a cell
  • labeling a bioparticle such as a cell with an antibody or a dye (particularly a fluorochrome) it is also possible to determine whether the bioparticle is a particle to be collected on the basis of a feature of a surface antigen of the bioparticle.
  • the detection unit 192 may acquire a bright field image and/or a phase difference image generated by the light irradiation unit 191 irradiating light.
  • the light irradiation unit 191 may include a halogen lamp
  • the detection unit 192 may include a CCD or a CMOS.
  • a bioparticle is irradiated with light by the halogen lamp, such that the CCD or CMOS may acquire a bright field image and/or a phase difference image of the irradiated bioparticle.
  • the determination unit 195 included in the control unit 193 determines whether the bioparticle is a particle to be collected on the basis of the acquired bright field image and/or phase difference image. For example, it may be determined whether the bioparticle is a particle to be collected on the basis of one or a combination of two or more of a form, a size, and a color of the bioparticle (particularly the cell).
  • the detection unit 192 may acquire a dark field image generated by the light irradiation unit 191 irradiating light.
  • the light irradiation unit 191 may include a laser light source
  • the detection unit 192 may include a CCD or a CMOS.
  • a bioparticle is irradiated with light by the laser, such that the CCD or CMOS may acquire a dark field image (e.g., a fluorescence image) of the irradiated microparticle.
  • the determination unit 195 included in the control unit 193 determines whether the bioparticle is a particle to be collected on the basis of the acquired dark field image. For example, it may be determined whether the bioparticle is a particle to be collected on the basis of one or a combination of two or more of a form, a size, and a color of the bioparticle (particularly the cell).
  • the detection unit 192 may be, for example, an imaging element in which a substrate equipped with a CMOS sensor and a substrate equipped with a digital signal processor (DSP) are laminated.
  • DSP digital signal processor
  • the imaging element can operate as a so-called AI sensor.
  • the detection unit 192 including the imaging element may determine whether the bioparticle is a particle to be collected, for example, on the basis of a learning model.
  • the learning model may be updated in real time while the method according to the present technology is being performed.
  • the DSP may perform machine learning processing while resetting a pixel array unit of the CMOS sensor, while exposing the pixel array unit, or while reading out a pixel signal from each unit pixel of the pixel array unit.
  • An example of the imaging element operating as an AI sensor may include an imaging device described in International Patent Application Publication No. 2018/051809. In a case where the AI sensor is used as an imaging element, raw data acquired from an image array is learned as it is, and thus, sorting-related determination processing is performed at a fast speed.
  • the determination may be performed, for example, based on whether the information regarding the feature of the light satisfies a criterion specified in advance.
  • the criterion may be a criterion indicating that the bioparticle is a particle to be collected.
  • the criterion may be appropriately set by a person skilled in the art, and may be, for example, a criterion related to a feature of light, such as a criterion used in the technical field pertaining to flow cytometry or the like.
  • Light may be irradiated toward one position in the detection region 156 , or light may be irradiated toward each of a plurality of positions in the detection region 156 .
  • the microchip 150 may be configured so that light is irradiated toward each of two different positions in the detection region 156 (that is, there are two positions irradiated with light in the detection region 156 ).
  • it may be determined whether the bioparticle is a particle to be collected, for example, on the basis of light (e.g., fluorescence and/or scattered light) generated by irradiating the bioparticle with light at one position.
  • a velocity of the bioparticle in the channel can be calculated on the basis of a difference between a time at which light generated by irradiating the light at one position is detected and a time at which light generated by irradiating the light at the other position is detected.
  • a distance between the two irradiation positions may be determined in advance, and the velocity of the bioparticle may be determined on the basis of the difference between the two detection times and the distance.
  • a difference between a time at which a certain bioparticle arrives at the particle sorting part 157 and a time at which a bioparticle before or after the certain bioparticle arrives at the particle sorting part 157 is equal to or smaller than a predetermined threshold value, it can be determined that the certain bioparticle is not collected.
  • a distance between a certain bioparticle and a bioparticle before or after the certain bioparticle is small, it is highly likely that the microparticle before or after the certain bioparticle is collected together with the certain bioparticle when the certain bioparticle is sucked.
  • control unit 193 may control irradiation of light by the light irradiation unit 191 and/or detection of light by the detection unit 192 . Furthermore, the control unit 193 may control driving of a pump for supplying a fluid into the microchip 150 .
  • the control unit 193 may include, for example, a hard disk in which a program for causing the device to execute the isolation step and an OS are stored, a CPU, and a memory.
  • the functions of the control unit 193 may be realized in a general-purpose computer.
  • the program may be recorded in a recording medium such as a micro SD memory card, an SD memory card, or a flash memory.
  • the program recorded in the recording medium may be read out by a drive (not illustrated) provided in the bioparticle sorting device 200 , and then the control unit 193 may cause the bioparticle sorting device 200 to execute the isolation step according to the read-out program.
  • the bioparticle determined as a particle to be collected in the determination step S 202 is collected into the collection channel 159 .
  • the particle to be collected is collected, while being contained in a first liquid, into a second liquid immiscible with the first liquid in the collection channel.
  • an emulsion containing the second liquid as a dispersion medium and the first liquid as a dispersoid can be formed in the collection channel 159 , with one particle to be collected being contained in each emulsion particle of the emulsion. This makes it possible to isolate an intended bioparticle into a space in an emulsion particle.
  • a particle P to be collected is collected, while being contained in the first liquid indicated by white, in the second liquid indicated by gray in FIG. 16 .
  • emulsion particles 190 are formed, and one particle P to be collected is isolated into a space in one emulsion particle 190 .
  • the collection step S 203 is performed in the particle sorting part 157 of the microchip 150 .
  • the laminar flow through the main channel 155 is divided into two waste channels 158 .
  • the particle sorting part 157 illustrated in FIG. 8 A has two waste channels 158 , but the number of branch channels is not limited to two.
  • the particle sorting part 157 may have one or a plurality of branched channels (e.g., two, three, or four branched channels).
  • the branch channels may be configured to branch in a Y shape on one plane as in FIG. 8 A , or may be configured to branch three-dimensionally.
  • FIG. 9 Only when a particle to be collected flow into the particle sorting part 157 , a flow from the main channel 155 into the collection channel 159 through the connection channel 170 is formed, such that the particle to be collected is collected into the collection channel 159 .
  • the enlarged view of the particle sorting part 157 is illustrated in FIG. 9 .
  • the main channel 155 and the collection channel 159 communicate with each other via the connection channel 170 that is coaxial with the main channel 155 .
  • the particle to be collected flows into the collection channel 159 through the connection channel 170 .
  • FIG. 9 C microparticles that are not particles to be collected flows into the waste channels 158 .
  • FIGS. 12 A and 12 B are enlarged views of the vicinity of the connection channel 170 .
  • FIG. 12 A is a schematic perspective view of the vicinity of the connection channel 170 .
  • FIG. 12 B is a schematic cross-sectional view on a plane passing through a central line of the liquid supply channel 161 and a central line of the connection channel 170 .
  • the connection channel 170 includes a channel 170 a close to the detection region 156 (hereinafter, also referred to as upstream connection channel 170 a ), a channel 170 b close to the collection channel 159 (hereinafter, also referred to as downstream connection channel 170 b ), and a connection portion 170 c between the connection channel 170 and the liquid supply channel 161 .
  • the liquid supply channel 161 is provided to be substantially perpendicular to an axis of the connection channel 170 .
  • the two liquid supply channels 161 are provided to face each other at a substantially central position of the connection channel 170 , but only one liquid supply channel may be provided.
  • a shape and a dimension of a cross section of the upstream connection channel 170 a may be the same as a shape and a dimension of a cross section of the downstream connection channel 170 b .
  • both the cross section of the upstream connection channel 120 a and the cross section of the downstream connection channel 120 b may be substantially circular while having the same dimension.
  • both of these two cross sections may be quadrilateral (e.g., square or rectangular), while having the same dimension.
  • the second liquid is supplied from the two liquid supply channels 161 to the connection channel 170 as indicated by arrows in FIG. 12 B .
  • the second liquid flows from the connection portion 170 c to both the upstream connection channel 170 a and the downstream connection channel 170 b.
  • the second liquid flows as follows.
  • the second liquid flowing into the upstream connection channel 170 a comes out of a surface of the connection channel 170 connected to the main channel 155 , and then flows dividedly into the two waste channels 158 . Since the second liquid comes out of the connected surface as described above, it is possible to prevent the first liquid and microparticles that do not need to be collected into the collection channel 159 from entering the collection channel 159 through the connection channel 170 .
  • the second liquid flowing to the downstream connection channel 170 b flows into the collection channel 159 .
  • the collection channel 159 is filled with the second liquid, and the second liquid serves as a dispersion medium, for example, for forming an emulsion.
  • the second liquid may be supplied from the two liquid supply channels 161 to the connection channel 170 .
  • a flow from the main channel 155 to the collection channel 159 through the connection channel 170 is formed. That is, a flow from the main channel 155 to the collection channel 159 through the upstream connection channel 170 a , the connection portion 170 c , and the downstream connection channel 170 b in this order is formed.
  • the particle to be collected is collected, while being surrounded by the first liquid, into the second liquid in the collection channel 159 .
  • an emulsion may be formed in the collection channel 159 or in a container connected to a collection channel end 163 , for example, via a channel.
  • a shape and/or a dimension of a cross section of the upstream connection channel 120 a may be different from a shape and/or a dimension of a cross section of the downstream connection channel 120 b .
  • FIGS. 13 A and 13 B illustrate an example in which these two channels have different dimensions.
  • a connection channel 180 includes a channel 180 a close to the detection region 156 (hereinafter, also referred to as upstream connection channel 180 a ), a channel 180 b close to the collection channel 159 (hereinafter, also referred to as downstream connection channel 180 b ), and a connection portion 180 c between the connection channel 180 and the liquid supply channel 161 .
  • Both a cross section of the upstream connection channel 180 a and a cross section of the downstream connection channel 180 b have a substantially circular shape, but the cross section of the latter has a larger diameter than the cross section of the former.
  • the cross section of the latter has a larger area than the cross section of the former, thereby making it possible to more effectively prevent already-collected microparticles from being discharged to the main channel 155 through the connection channel 180 as described above.
  • a particle to be collected is collected into the collection channel through the connection channel due to a change in pressure in the collection channel 159 .
  • the collection may be performed, for example, by generating a negative pressure in the collection channel 159 as described above.
  • the negative pressure may be generated when a wall defining the collection channel 159 is deformed, for example, by an actuator 197 (particularly a piezoelectric actuator) attached to the outside of the microchip 150 .
  • the flow entering the collection channel 159 may be formed by the negative pressure.
  • the actuator 197 may be attached to the outside of the microchip 150 so that the wall of the collection channel 159 may be deformed.
  • the deformation of the wall makes it possible to change an inner space of the collection channel 159 and generate a negative pressure.
  • the actuator 197 may be, for example, a piezoelectric actuator.
  • the particle to be collected is collected, while being surrounded by the first liquid, into the second liquid immiscible with the first liquid in the collection channel 159 .
  • an emulsion containing the second liquid as a dispersion medium and the first liquid as a dispersoid is formed in the collection channel 159 as described above.
  • connection channel 170 is provided with the liquid supply channel 161 .
  • the second liquid immiscible with the liquid (the sample liquid and the sheath liquid) flowing through the main channel 155 is introduced from the liquid supply channel 161 into the connection channel 170 .
  • a flow from the connection channel 170 toward the main channel 155 is formed by a part of the second liquid introduced into the connection channel 170 to prevent bioparticles other than the particles to be collected from entering the collection channel 159 .
  • the second liquid flowing from the connection channel 170 toward the main channel 155 flows through the waste channels 158 similarly to the first liquid, rather than flowing into the main channel 155 , due to the flow of the first liquid to the waste channels 158 through the main channel 155 .
  • the rest of the second liquid introduced into the connection channel 170 flows into the collection channel 159 .
  • the collection channel 159 may be filled with the second liquid.
  • the collection channel 159 may be filled with the second liquid immiscible with the first liquid.
  • the second liquid may be supplied from the liquid supply channel 161 to the connection channel 170 .
  • the second liquid flows from the connection channel 170 to the collection channel 159 , and accordingly, the collection channel 159 may be filled with the second liquid.
  • the liquid flowing into the waste channels 158 in laminar flow may be discharged out of the microchip at waste channel ends 160 .
  • the target particle collected into the collection channel 159 may be discharged out of the microchip at the collection channel end 161 .
  • a container 171 may be connected to the collection channel end 163 via a channel such as a tube 172 .
  • a channel such as a tube 172 .
  • an emulsion containing the first liquid containing a particle to be collected as a dispersoid and the second liquid as a dispersion medium is collected into the container 171 .
  • the bioparticle sorting device 200 may include a channel for collecting an emulsion containing a particle to be collected into the container.
  • a plurality of emulsion particles can be held in the collection channel 159 .
  • an assay such as single cell analysis can be continuously performed in the collection channel 159 .
  • the disruption step to be described later may be performed in the collection channel 159 .
  • a target substance may be bound to a target capturing molecule in the disruption step.
  • the main channel may be branched into the connection channel and the at least one waste channel.
  • the at least one waste channel is a channel through which bioparticles other than particles to be collected flow.
  • the main channel, the connection channel, and the collection channel may be arranged in a straight line.
  • the collection step can be performed more efficiently as compared with, for example, a case where the connection channel and the collection channel are arranged at an angle with respect to the main channel. For example, a suction amount required to guide a particle to be collected to the connection channel can be reduced.
  • bioparticles flow toward the connection channel while being arranged substantially in a line in the main channel. Therefore, a suction amount can be reduced in the collection step.
  • FIG. 16 illustrates a microchip in which inlets and outlets are formed as described above.
  • all of the collection channel end 163 and the two branch channel ends 160 are formed on a surface where the sample liquid inlet 151 and the sheath liquid inlet 153 are formed.
  • an introduction channel inlet 164 for introducing liquid into the introduction channel 161 is formed in the surface.
  • the microchip 250 for sorting bioparticles all of the inlets into which the liquids are introduced and the outlets from which the liquids are discharged are formed in one surface.
  • a part of the sheath liquid channel 154 is indicated by a dotted line.
  • the portion indicated by the dotted line is disposed at a lower position than the sample liquid channel 152 indicated by a solid line (a position shifted in an optical axis direction indicated by an arrow), and these channels do not communicate with each other at a position where the channel indicated by the dotted line and the channel indicated by the solid line intersect.
  • This description also applies to a part of the collection channel 159 indicated by a dotted line and the branch channel 158 intersecting the part of the collection channel 159 .
  • the liquid supply channel supplies a liquid (particularly the second liquid) to the connection channel.
  • a flow from a position at which the liquid supply channel and the connection channel are connected to each other toward the main channel is formed in the connection channel, thereby making it possible to prevent the liquid flowing through the main channel from entering the connection channel and prevent microparticles other than particles to be collected from flowing into the collection channel through the connection channel.
  • the collection step is performed, the first liquid containing one particle to be collected is collected into the second liquid in the collection channel through the connection channel, for example, due to a negative pressure generated in the collection channel, as described above. As a result, an emulsion particle containing one particle to be collected is formed in the second liquid.
  • a hydrophobic solution including a bioparticle determined to be a particle to be collected in the determination step is collected into the collection channel 159 , for example, by driving the piezoelectric actuator at an appropriate timing (for example, at a time when the bioparticle arrives at the particle sorting part 157 ), to form an emulsion particle.
  • the determination step by determining whether the particle is a particle to be collected, for example, using a peak signal and an area signal, it is also possible to determine whether there is one microparticle (singlet), a conjugate of two bioparticles (doublet), or a conjugate of three bioparticles (triplet).
  • an emulsion particle containing one bioparticle can be formed with high probability and high efficiency. Furthermore, since it is possible to avoid forming an emulsion particle containing a conjugate of two or more bioparticles as described above, it is possible to omit an operation of removing a conjugate of two or more bioparticles before an operation of forming an emulsion, for example, by a cell sorter or the like.
  • an emulsion particle containing one microparticle was formed as follows. Hereinafter, an operation for forming an emulsion particle will be described with reference to FIG. 15 .
  • a piezoelectric element (a piezoelectric actuator) was attached to an outer surface of the microchip 150 (particularly an outer surface corresponding to a bulging portion close to a portion of the collection channel 159 connected to the connection channel 170 ) to change a volume in the collection channel 159 .
  • a hydrophilic sample liquid containing beads having a diameter of 10 ⁇ m was introduced from the sample liquid inlet 151 into the sample liquid channel 152 , and a hydrophilic sheath liquid was introduced from the sheath liquid inlet 153 into the sheath liquid channel 154 .
  • the hydrophilic sample liquid and the hydrophilic sheath liquid introduced joined together at the junction portion 162 , and then, a laminar flow in which the hydrophilic sample liquid was surrounded by the hydrophilic sheath liquid was formed.
  • the laminar flow included beads arranged substantially in a line, and the laminar flow was formed in the main channel 155 toward the connection channel 170 .
  • a hydrophobic liquid was supplied from the liquid supply channel 161 to the connection channel 170 .
  • the hydrophobic liquid was supplied from the liquid supply channel 161 to the connection channel 170 .
  • the piezoelectric element was driven to deform an inner cavity of the collection channel 159 , and accordingly, the bead was collected into the collection channel 159 through the connection channel 170 .
  • the operations for the collection were as follows: (i) the collection channel 159 was deformed over 10 ⁇ s to apply a negative pressure into the collection channel 159 , (ii) the deformed state was maintained for 10 ⁇ s, and (iii) the negative pressure was eliminated over 10 ⁇ s to restore the deformation.
  • an emulsion containing emulsion particles each containing one bead in the hydrophobic liquid was formed in the collection channel 159 .
  • the hydrophobic liquid was a dispersion medium and the emulsion particle containing the hydrophilic liquid was a dispersoid.
  • FIG. 15 It is shown in FIG. 15 that an emulsion particle containing one bead is formed in the collection channel 159 by performing the above-described operations (i) to (iii). FIG. 15 will be described below.
  • FIG. 15 is a photograph showing a state in which the bead (indicated by a white arrow) flows toward the connection channel 170 .
  • the hydrophilic liquid flows in the main channel 155 toward the particle sorting part 157 , and then flows to the two waste channels 158 branched from the main channel 155 without flowing to the connection channel 170 .
  • the hydrophobic liquid is supplied from the liquid supply channel 161 to the connection channel 170 , and flows to both the main channel 155 and the collection channel 159 .
  • the hydrophobic liquid having flowed into the main channel 155 flows dividedly into the two waste channels 158 immediately after leaving the connection channel 170 due to the flow of the hydrophilic liquid (the hydrophobic liquid flows along walls of the waste channels 158 in the vicinity of the connection channel shown in a of FIG. 15 ).
  • FIG. 15 is a photograph at a time point when the bead further approaches the connection channel. At this time point, an operation for collecting the bead is started. That is, deforming the inner cavity of the collection channel 159 is started by driving the piezoelectric element.
  • FIG. 15 is a photograph showing a state in which the hydrophilic liquid is advancing toward the collection channel 159 . It can be seen from the photograph that, at a time point of FIG. 15 , the bead is surrounded by the hydrophilic liquid, and the hydrophilic liquid is surrounded by the hydrophobic liquid. That is, an emulsion particle P containing one bead is formed.
  • FIG. 15 is a photograph immediately before the emulsion particle P enters the collection channel 159 from the connection channel 170 .
  • FIG. 15 is a photograph immediately after the emulsion particle P enters the collection channel 159 . It can be confirmed that one bead is contained in the emulsion particle P.
  • FIG. 15 is a photograph showing that the emulsion particle P has flowed further downstream in the marine channel 159 .
  • the bioparticle sorting device 200 can form an emulsion containing a high proportion of emulsion particles each containing one bioparticle.
  • the bioparticle is disrupted in the microspace.
  • the disruption for example, the molecule 100 bound to the bioparticle via the bioparticle capturing part 7 may be dissociated from the bioparticle.
  • a component bound to the bioparticle capturing part 7 may be bound to the molecule 100 via the bioparticle capturing part 7 even after the disruption.
  • a target substance constituting the bioparticle or a target substance bound to the bioparticle may be captured by the target substance capturing part 6 included in the molecule 100 .
  • a complex of the molecule 100 and the target substance is formed, such that the target substance can be associated with a barcode sequence included in the molecule 100 in the analysis step to be described later.
  • the complex formed in this way is analyzed in the analysis step to be described later.
  • the disruption step S 105 is executed preferably while the isolated state of the bioparticle in the microspace is maintained. As a result, a complex of the molecule 100 and the target substance is efficiently formed. Furthermore, it is possible to prevent the target substance from being bound to a target capturing molecule outside the microspace.
  • the maintaining of the isolated state may mean that the emulsion particle is maintained, and particularly, may mean that the emulsion particle is not disrupted.
  • the maintaining of the isolated state may mean that a component in the well (particularly a bioparticle in the well, a component of the bioparticle, and a target capturing molecule) remains in the well, and may further mean that a component in another well (particularly a bioparticle in another well, a component of the bioparticle, and a target capturing molecule in another well) does not invade the well.
  • nucleic acid-bound antibody when a nucleic acid-bound antibody is bound to a bioparticle as described in the section “(3-2) Capture Step” above, the nucleic acid-bound antibody is dissociated from the bioparticle in the disruption step S 105 . Then, the nucleic acid-bound antibody is bound to the target substance, such that a complex of the nucleic acid-bound antibody and the target substance may be formed.
  • a poly A sequence constituting the first nucleic acid 21 may be bound to an mRNA in a bioparticle as a target substance. Since the second nucleic acid 2 including an antibody barcode sequence is bound to the first nucleic acid 21 , the target substance can be associated with the antibody barcode sequence. The complex formed in this way is analyzed in the analysis step to be described later.
  • the disruption step S 105 may be performed by chemically or physically disrupting the bioparticle.
  • a bioparticle-disrupting substance may be brought into contact with the bioparticle in the microspace.
  • the bioparticle-disrupting substance may be appropriately selected by a person skilled in the art depending on the type of bioparticle.
  • a lipid bilayer membrane disrupting component may be used as the bioparticle disrupting substance, and particularly, a surfactant, an alkali component, an enzyme, or the like may be used.
  • a surfactant an anionic surfactant, a nonionic surfactant, an amphoteric surfactant, or a cationic surfactant may be used.
  • Examples of the anionic surfactant may include sodium dodecyl sulfate (SDS) and sodium lauroyl sarcosine.
  • Examples of the nonionic surfactant may include Triton X-100, Triton X-114, Tween 20, Tween 80, NP-40, Brij-35, Brij-58, octyl glucoside, octylthioglucoside, and octylphenoxypolyethoxyethanol.
  • Examples of the amphoteric surfactant may include CHAPS and CHAPSO.
  • Examples of the cationic surfactant may include cetyltrimethylammonium bromide (CTAB).
  • examples of the alkali component may include OH ⁇ ions.
  • examples of the enzyme may include proteinase K, streptolidine, lysozyme, lysostaphin, zymolase, cellulase, glycanase, and protease.
  • the type of enzyme may be appropriately selected, for example, depending on the type of cell (animal cell, plant cell, bacteria, yeast, or the like).
  • the disruption step may be performed, for example, by adding a bioparticle-disrupting substance to each well. Since the wells are isolated from each other, the components in the wells are maintained in the wells even though disruption occurs.
  • a bioparticle-disrupting substance may be introduced into the emulsion particle simultaneously with the formation of the emulsion particle. Then, after the emulsion particle is formed, a step of disrupting the bioparticle by the bioparticle-disrupting substance may be performed.
  • a treatment for applying the physical stimulation to the bioparticle for example, an optical treatment, a thermal treatment, an electrical treatment, an acoustic treatment, a freeze-thaw treatment, or a mechanical treatment may be adopted. Through such a treatment, a cell or an exosome can be disrupted.
  • the optical treatment may include plasma formation or cavitation bubble formation by laser light irradiation.
  • the thermal treatment may include a heat treatment.
  • the acoustic treatment may include sonication using ultrasonic waves.
  • the mechanical treatment may include a treatment using a homogenizer or a bead mill.
  • the physical disruption of the bioparticle through such a treatment can be applied to both the case where the microspace is a space in a well and the case where the microspace is a space in an emulsion particle.
  • an optical treatment, a thermal treatment, an electrical treatment, and a freeze-thaw treatment are particularly suitable among the above-described treatments.
  • a surfactant may be added into the emulsion particle, and moreover, a concentration of the surfactant may be adjusted.
  • the collection sequence part 2 included in the molecule 100 may be used.
  • a target substance may be bound to the molecule 100 , and the target substance can be efficiently collected by using the collection sequence part 2 . That is, the disruption step S 105 includes a step of collecting the molecule 100 (particularly the target substance bound to the molecule 100 ) using the collection sequence part 2 .
  • a bioparticle is analyzed.
  • a target substance is analyzed.
  • An analysis method may be determined, for example, depending on the type of target substance and the purpose of analysis.
  • the barcode sequence and the target substance may be associated with each other. More specifically, the barcode sequence may be associated with a result of analyzing the target substance.
  • the analysis result may include, for example, sequence information and/or amount information about the target substance.
  • one bioparticle is isolated into one microspace, and a plurality of molecules 100 capturing the one bioparticle all have the same barcode sequence. Therefore, all analysis results associated with one barcode sequence by performing the above-described association are derived from one bioparticle, and can be used for analyzing the one bioparticle.
  • the analysis step S 106 since a molecule 100 including a barcode sequence is bound to a target substance in the disruption step, even when different bioparticles present in a plurality of microspaces are collectively analyzed, an analysis result can be associated with each bioparticle on the basis of the barcode sequence.
  • each bioparticle disruption product in the well may be separately analyzed, or bioparticle disruption products in a plurality of wells may be collected as one sample, and collectively analyzed as the one sample.
  • the former it is easy to associate a bioparticle with an analysis result.
  • the latter since a target substance in each bioparticle disruption product forms a complex with a molecule 100 including a barcode sequence (or a nucleic acid-bound antibody including an antibody barcode sequence), each bioparticle can be associated with an analysis result thereof.
  • a plurality of emulsion particles may be collectively analyzed, and for example, the obtained emulsion may be collectively analyzed in its entirety. Since a target substance in each bioparticle disruption product forms a complex with a molecule 100 including a barcode sequence (or a nucleic acid-bound antibody including an antibody barcode sequence), each bioparticle can be associated with an analysis result thereof. As a result, analysis efficiency can be improved.
  • a bioparticle is analyzed.
  • the analysis may be performed, for example, on a complex of a molecule 100 and a target substance and/or a complex of a nucleic acid-bound antibody and a target substance formed in the disruption step S 105 . Since the molecule 100 and the nucleic acid-bound antibody include a barcode sequence and an antibody barcode sequence, respectively, it is possible to identify a bioparticle from which the target substance is derived on the basis of the barcode sequence.
  • the target substance has a base sequence
  • the target substance is an RNA (particularly an mRNA) or a DNA
  • the base sequence of the target substance may be subjected to sequencing processing in the analysis step S 106 .
  • the sequencing processing may be performed, for example, by a next-generation sequencer.
  • the analysis in the analysis step S 106 may be performed, for example, using the amplification sequence part 3 included in the molecule 100 .
  • the analysis step S 106 includes a step of amplifying a nucleic acid using the amplification sequence part 3 .
  • a nucleic acid (particularly an mRNA) bound to the molecule 100 may be amplified.
  • information regarding the nucleic acid can be acquired by performing sequencing processing on the sequence of the nucleic acid.
  • the barcode sequence included in the barcode sequence part may also be amplified.
  • the information regarding the nucleic acid can be associated with the barcode sequence included in the molecule 100 , and further, can be associated with the bioparticle.
  • the analysis step S 106 may be performed using an analysis device 120 as illustrated in FIG. 3 F .
  • the analysis device 120 may be, for example, a device that performs sequencing processing on the complex.
  • the sequencing processing may be performed, for example, in a case where the target substance is a nucleic acid, particularly a DNA or an RNA, and more particularly an mRNA.
  • the sequencing processing may be performed by a sequencer, or may be performed by a next-generation sequencer or a Sanger method-based sequencer. In order to comprehensively analyze a plurality of bioparticles (particularly a cell population) at a higher speed, the sequencing processing may be performed by a next-generation sequencer.
  • the analysis step may further include a step of preparing a nucleic acid (e.g., a cDNA) to be subjected to sequencing processing and a step of purifying the nucleic acid.
  • a nucleic acid e.g., a cDNA
  • the analysis step may further include a step of preparing a nucleic acid (e.g., a cDNA) to be subjected to sequencing processing and a step of purifying the nucleic acid.
  • a nucleic acid e.g., a cDNA
  • the collection sequence part 2 may be used.
  • a molecule 100 to which a target substance is bound may be collected using a bead on which a nucleic acid having a sequence complementary to the nucleic acid sequence included in the collection sequence part 2 is immobilized.
  • the preparation step may include, for example, a cDNA synthesis step of synthesizing a cDNA from an mRNA. Furthermore, the preparation step may include an amplification step of amplifying the synthesized cDNA.
  • a purification step of purifying the nucleic acid obtained in the preparation step may be performed.
  • the purification step may include, for example, a treatment for degrading components other than the nucleic acid using an enzyme such as proteinase K.
  • a nucleic acid collection treatment may be performed.
  • a commercially available reagent for purifying nucleic acids may be used, and examples thereof may include magnetic beads such as AMPure XP. Note that, in the purification step, an intracellular dsDNA can also be collected, but the dsDNA can be prevented from being sequenced in the sequencing processing.
  • an adaptor sequence for sequencing processing (particularly for next-generation sequencing processing) is included in a target capturing molecule, thereby making it possible to sequence only a nucleic acid including the adaptor sequence.
  • components may be analyzed for each bioparticle on the basis of a sequencing processing result. For example, in the analysis step S 106 , a sequence of an mRNA included in a cell and/or the number of copies of each mRNA may be determined for each bioparticle. Furthermore, in the analysis step S 106 , the type and/or the number of antigens or the type and/or the number of transcription factors may be determined for each bioparticle.
  • Such analysis of the components for each bioparticle may be performed on the basis of a barcode sequence among sequences determined by the sequence processing. For example, sequences including the same barcode sequence are selected from among a large number of sequences determined by the sequence processing. The sequences including the same barcode sequence are based on target capturing molecules taken up by one cell. Therefore, the analysis of the components for each barcode sequence means that components are analyzed for each bioparticle.
  • the present technology also provides a bioparticle analysis system including a capture kit for capturing a bioparticle, the capture kit having a surface on which a molecule including a bioparticle capturing part, a barcode sequence, and a cleavable linker is immobilized via the linker.
  • the bioparticle analysis system may further include a linker cleavage device that cleaves the linker to release the bioparticle from the surface, and/or an isolation device that forms or has a microspace into which the bioparticle released from the surface is isolated.
  • the capture kit may include, for example, a substrate having a surface on which a molecule including a bioparticle capturing part, a barcode sequence, and a cleavable linker is immobilized via the linker.
  • the substrate may be an analysis substrate 102 as described in section (3-1) of “1. First Embodiment (Bioparticle Analysis Method)” above, and the description thereof also applies to the present embodiment.
  • the linker cleavage device may be, for example, a stimulation application device as described in section (3-3-2) of “1. First Embodiment (Bioparticle Analysis Method)” above, and the description thereof also applies to the present embodiment.
  • the bioparticle analysis system may further include an imaging element that images the surface 101 or the bioparticle captured on the surface 101 .
  • the linker cleavage device may apply stimulation so that a bioparticle selected on the basis of an image acquired by the imaging element is dissociated from the surface 101 .
  • the isolation device may include a well as described in section (3-4-3) of “1. First Embodiment (Bioparticle Analysis Method)” above, and may include, for example, a substrate (e.g., a plate) including the well. Furthermore, the isolation device may include a nozzle that applies a bioparticle-containing droplet to the well as described in section (3-4-3) above.
  • the isolation device may include a device that puts one bioparticle in one well, such as a cell sorter or a single cell dispenser, as described in section (3-4-3) above.
  • the isolation device may include a microchannel as described in section (3-4-4) of “1. First Embodiment (Bioparticle Analysis Method)” above, or may include a microchip 150 as described in section (3-4-4) above.
  • the isolation device may be a bioparticle sorting device 200 as described in section (3-4-4) above.
  • the bioparticle analysis system may further include an analysis device that analyzes a target substance constituting the bioparticle or a target substance bound to the bioparticle.
  • the analysis device may be a device that performs analysis as described in section (3-6) of “1. First Embodiment (Bioparticle Analysis Method)” above, e.g., a sequencer.
  • the bioparticle analysis system according to the present technology may perform the bioparticle analysis method described in “1. First Embodiment (Bioparticle Analysis Method)” above. Therefore, the description regarding the bioparticle analysis method also applies to the present embodiment.
  • the bioparticle analysis system according to the present technology may perform a capture step S 102 , a cleavage step S 103 , and an isolation step S 104 as described above. Furthermore, the bioparticle analysis system according to the present technology may perform a disruption step S 105 after the isolation step S 104 , and may further perform a part of an analysis step S 106 , for example, a sample preparation step of preparing a sample to be subjected to the analysis step S 106 , after the disruption step S 105 .
  • the sample preparation step may include a cDNA synthesis step of synthesizing a cDNA from an mRNA.
  • the system may be configured to automatically perform the capture step S 102 , the cleavage step S 103 , and the isolation step S 104 , may particularly be configured to automatically perform the capture step S 102 , the cleavage step S 103 , the isolation step S 104 , and the disruption step S 105 , and may more particularly be configured to automatically perform the capture step S 102 , the cleavage step S 103 , the isolation step S 104 , the disruption step S 105 , and the analysis step S 106 (or the sample preparation step or the like as a part thereof).
  • the present technology also provides a surface on which a molecule including a bioparticle capturing part, a barcode sequence, and a cleavable linker is immobilized via the linker.
  • the surface is a surface as described in “1. First Embodiment (Bioparticle Analysis Method)” above, and the description thereof also applies to the present embodiment.
  • the present technology also provides a substrate having the surface. The surface and the substrate may be those used in “1. First Embodiment (Bioparticle Analysis Method)” above.
  • a bioparticle analysis method including:
  • bioparticle analysis method in which in the capture step, a plurality of molecules bound to one bioparticle have the same barcode sequence.
  • bioparticle analysis method according to [1] or [2], further including a disruption step of disrupting the bioparticle in the microspace.
  • a target substance constituting the bioparticle or a target substance bound to the bioparticle is captured by the target substance capturing part.
  • bioparticle analysis method according to [5], further including an analysis step of analyzing the target substance after the disruption step.
  • bioparticle analysis method in which in the cleavage step, a bioparticle to be released from the surface is selected on the basis of a label of the bioparticle or a label of the molecule.
  • bioparticle analysis method according to any one of [1] to [10], in which in the cleavage step, the captured state of the bioparticle is maintained by the bioparticle capturing part.
  • bioparticle analysis method according to any one of [1] to [11], in which in the cleavage step, only a selected bioparticle is released from the surface.
  • bioparticle analysis method according to any one of [1] to [12], in which the microspace is a space in an emulsion particle or a space in a well.
  • bioparticle analysis method according to any one of [1] to [13], further including a determination step of determining whether to isolate a bioparticle released from the surface in the cleavage step into a microspace.
  • bioparticle analysis method according to any one of [1] to [15], in which in the capture step, the bioparticle and the bioparticle capturing part are bound to each other in a specific or non-specific manner.
  • bioparticle analysis method according to any one of [1] to [16], in which the capture step includes an incubation step for binding the bioparticle and the bioparticle capturing part to each other.
  • a bioparticle analysis system including:
  • a capture kit configured to capture a bioparticle, the capture kit having a surface on which a molecule including a bioparticle capturing part, a barcode sequence, and a cleavable linker is immobilized via the linker;
  • linker cleavage device that cleaves the linker to release the bioparticle from the surface
  • an isolation device that forms or has a microspace into which the bioparticle released from the surface is isolated.
  • the bioparticle analysis system according to [18], further including an analysis device that analyzes a target substance constituting the bioparticle or a target substance bound to the bioparticle.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Organic Chemistry (AREA)
  • Engineering & Computer Science (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Immunology (AREA)
  • Molecular Biology (AREA)
  • Biochemistry (AREA)
  • Biotechnology (AREA)
  • Analytical Chemistry (AREA)
  • Physics & Mathematics (AREA)
  • Microbiology (AREA)
  • General Health & Medical Sciences (AREA)
  • Biophysics (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • Genetics & Genomics (AREA)
  • Biomedical Technology (AREA)
  • Urology & Nephrology (AREA)
  • Hematology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Cell Biology (AREA)
  • Virology (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Food Science & Technology (AREA)
  • Medicinal Chemistry (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
US18/012,605 2020-07-06 2021-06-17 Bioparticle analysis method and bioparticle analysis system Pending US20230242983A1 (en)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
JP2020116432 2020-07-06
JP2020-116432 2020-07-06
PCT/JP2021/023036 WO2022009642A1 (fr) 2020-07-06 2021-06-17 Système et procédé d'analyse de bioparticules

Publications (1)

Publication Number Publication Date
US20230242983A1 true US20230242983A1 (en) 2023-08-03

Family

ID=79552986

Family Applications (1)

Application Number Title Priority Date Filing Date
US18/012,605 Pending US20230242983A1 (en) 2020-07-06 2021-06-17 Bioparticle analysis method and bioparticle analysis system

Country Status (5)

Country Link
US (1) US20230242983A1 (fr)
EP (1) EP4177353A4 (fr)
JP (1) JPWO2022009642A1 (fr)
CN (1) CN115768863A (fr)
WO (1) WO2022009642A1 (fr)

Families Citing this family (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2023188896A1 (fr) * 2022-03-29 2023-10-05 ソニーグループ株式会社 Système d'analyse de bioparticules, dispositif de traitement d'informations et procédé d'analyse de bioparticules
WO2024202267A1 (fr) 2023-03-30 2024-10-03 Sony Group Corporation Procédé d'analyse par formation d'acide nucléique circulaire
WO2024204517A1 (fr) 2023-03-30 2024-10-03 Sony Group Corporation Procédé d'analyse, procédé de fabrication d'acide nucléique circulaire et acide nucléique

Family Cites Families (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2014072465A1 (fr) * 2012-11-09 2014-05-15 Roche Diagnostics Gmbh Capture et analyse in vitro de cellules tumorales circulantes
JP6186812B2 (ja) 2013-04-04 2017-08-30 ソニー株式会社 粒子分取装置及び粒子分取方法
KR102333255B1 (ko) * 2015-05-11 2021-12-01 일루미나, 인코포레이티드 치료제의 발견 및 분석을 위한 플랫폼
CA2993463A1 (fr) * 2015-07-27 2017-02-02 Illumina, Inc. Cartographie spatiale d'informations de sequence d'acide nucleique
CN109691079B (zh) 2016-09-16 2021-05-14 索尼半导体解决方案公司 成像装置和电子设备
FI3583214T3 (fi) * 2017-02-02 2023-12-19 New York Genome Center Inc Menetelmiä ja koostumuksia biologisen näytteen sisältämien kohteiden tunnistamiseksi tai kvantifioimiseksi

Also Published As

Publication number Publication date
CN115768863A (zh) 2023-03-07
WO2022009642A1 (fr) 2022-01-13
JPWO2022009642A1 (fr) 2022-01-13
EP4177353A1 (fr) 2023-05-10
EP4177353A4 (fr) 2024-01-17

Similar Documents

Publication Publication Date Title
US11667907B2 (en) Method and apparatus for encoding cellular spatial position information
US20230242983A1 (en) Bioparticle analysis method and bioparticle analysis system
US12103004B2 (en) Method and apparatus for processing tissue and other samples encoding cellular spatial position information
EP3248018B1 (fr) Dispositifs et systèmes permettant le codage moléculaire à barres d'acides nucléiques cibles dans des cellules individuelles
US20240027311A1 (en) Method for processing tissue samples
KR102502083B1 (ko) 휴대용 핵산 분석 시스템 및 고성능 미세유체 전기활성 중합체 작동기
KR20080096567A (ko) 마이크로유체 장치
WO2021084814A1 (fr) Procédé de collecte de particules minuscules, micropuce pour aliquoter des particules minuscules, dispositif de collecte de particules minuscules, procédé de production d'émulsion, et émulsion
JP2024036647A (ja) 粒子確認方法、粒子捕捉用チップ、及び粒子分析システム
US20220106631A1 (en) Barcoding in droplets for cell-cell interaction and secreted protein detection and analysis
US20230066282A1 (en) Analysis method, analysis system, and analysis surface
US20240310375A1 (en) Bioparticle analysis method and reagent kit for bioparticle analysis
WO2023188896A1 (fr) Système d'analyse de bioparticules, dispositif de traitement d'informations et procédé d'analyse de bioparticules
WO2023168018A2 (fr) Procédé et appareil de traitement de tissu et d'autres échantillons codant des informations de position spatiale cellulaire avec codage combinatoire
Busk Fabrication and utilisation of solid-phase microdroplet arrays for nucleic acid detection and other applications
Cao Microfluidic Engineering for Ultrasensitive Molecular Analysis of cells

Legal Events

Date Code Title Description
AS Assignment

Owner name: SONY GROUP CORPORATION, JAPAN

Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNOR:MATSUMOTO, MASAHIRO;REEL/FRAME:063018/0363

Effective date: 20221117

STPP Information on status: patent application and granting procedure in general

Free format text: DOCKETED NEW CASE - READY FOR EXAMINATION